The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. The lyic replication cycle is comprised of rounds of host cell invasion, replication in the intracellular vacuole, and egress from the host cell. The essential host cell invasion step has been studied for its potential as a novel and specific drug target. During this process, sequential secretion of three secretory organelles (micronemes, rhoptries, and dense granules) occurs. Of these, it is the microneme proteins that contribute most to egress, gliding motility, and host cell invasion. These diverse functions have led to speculation as to whether there are micronemes with different contents secreted at different times, or whether microneme protein function is regulated by differences in parasite environment. Observations in support and against both models abound but there is no overwhelmingly convincing data to settle the argument in either direction. To contribute data that will inform this important debate, this proposal exploits the differential microneme secretion kinetics of two different conditional mutants in the Ca2+-dependent secretion machinery used by the micronemes. One mutant has a temperature sensitive (ts) mutation in TgDOC2 (ts-DOC2), and the other harbors a dominant negative allele of Ferlin-like protein TgFLP (DN-FLP) generated by conditional overexpression (ligand-controlled) of a TgFLP allele lacking the transmembrane domain. ts-DOC2 secretes no micronemes at all and will yield a background free of proteins secreted through the micronemes. DN-FLP has functional constitutive microneme secretion but is impaired in Ca2+-ionophore induced secretion. This mutant will facilitate differentiation between microneme proteins secreted at different points along the parasite's egress-motility-invasion journey. Thus, these data will contribute to the discussion of whether or not there is differential microneme protein secretion as outlined above. Excretory and secretory antigens (ESA) will be collected from mutant ts-DOC2 and DN-FLP grown under both the permissive and restrictive conditions. We will apply Stable Isotope Labeling by Amino Acids in Culture (SILAC). Most pertinent to the goals of this project, SILAC is quantitative and permits the detection of minor differences in protein abundance in complex mixtures across different mutants and under permissive versus restrictive conditions. Overall, the combination of different secretion mutants with the power of SILAC will result in an unprecedented level of ESA resolution and will either boost our confidence in, or disprove the possibility of differential microneme secretion. Hence, these will be valuable data sets for the Toxoplasma community in understanding the uniquely parasitic process of host cell invasion, and the insights could be of use in rational drug design.